PROJECT SUMMARY Type 2 diabetes mellitus (T2DM) and periodontitis are chronic diseases affecting millions of people globally and have long been considered biologically linked. Periodontitis is a complex inflammatory disease, initiated by subgingival microbial agents in a susceptible host, which can lead to tooth loss and contribute to systemic inflammation. Diabetes is an established risk factor for periodontitis. Importantly, periodontitis has also been shown to adversely affect glycemic control and to contribute to incident T2DM and the development of its complications. The innate immune system, including monocytes and macrophages, plays a pivotal role in the pathogenesis of both diseases. Functionally, macrophages can be categorized as classic pro-inflammatory macrophages (M1) or anti-inflammatory/resolution macrophages (M2). Our preliminary results indicate disruption of monocyte/macrophage homeostasis in periodontitis and T2DM and distinct RNA signature in cellular subtypes. We also found a decrease in JMJD3 and IRF4 in monocytes/macrophages of T2DM patients compared to subjects without T2DM. Downregulation of the JMJD3-IRF4 pathway has been linked to the suppression of M2 macrophage polarization. Taken together these findings indicate the need for a more comprehensive understanding of the role of monocyte/macrophage signaling in periodontitis with or without T2DM and for mechanistic studies to delineate perturbed pathways. In this proposal, in Aim 1, we will use single-cell RNA-sequencing and immune cell barcoding to first match the gene expression profile to the cellular sub-types of myeloid-derived immune cells in gingival tissue (locally) and immune cells in blood (systemically) with the vision of understanding their specific changes and their role in the pathogenesis of periodontitis in T2DM. Moreover, we will identify the molecular signaling events that affect the phenotype and function of monocytes/macrophages in the pathogenesis of periodontitis and T2DM. In Aim 2, We will use CRISPR/CAS9 activators to engineer monocytes/macrophages to restore JMJD3-IRF4 levels and modulate their functional profile ex-vivo towards a resolution (M2) phenotype. The results of the proposed studies will lead to an in-depth understanding of the heterogeneity in molecular signature and function of myeloid cells and their interactions and roles in the pathogenesis of periodontitis with and without T2DM and provide mechanistic tools to modulate macrophage phenotype to treat periodontitis with or without T2DM. The long-term vision of these studies is the development of actionable targeted molecular therapies that can modulate monocyte/macrophage phenotype and function in affected individuals. The proposed work is novel; it will inform future directions for in vivo studies and produce valuable data that will serve as preliminary work for an R01 grant application.